life Article Complete Mitochondrial Genome of Trichuris trichiura from Macaca sylvanus and Papio papio Julia Rivero , Rocío Callejón and Cristina Cutillas * Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Calle San Fernando, 4, 41004 Sevilla, Spain; [email protected] (J.R.); [email protected] (R.C.) * Correspondence: [email protected] Abstract: Trichuriasis is among the most prevalent worldwide parasitism caused by helminths. For many years, Trichuris spp. have been described with a relatively narrow range of both morphological and biometrical features. The use of the complete mitochondrial genome (mitogenome) is an alternative and powerful molecular method for inferring phylogenies. Here, we present an overview of the contributions of mitogenome for Trichuris spp. from human and non-human primates. In addition, we carry out structural and phylogenetic comparative analyses with genomes of Trichuris species available in public datasets. The complete mt genomes of Trichuris trichiura and Trichuris sp. from Macaca sylvanus and T. trichiura from Papio papio are 14,091 bp, 14,047 bp and 14,089 bp in length, respectively. The three mt genomes are circular and consist of 37 genes—13 PCGs (cox1–3, nad1–6, nad4L, atp6, atp8 and cob), 22 transfer RNA genes (tRNAs), and two rRNAs (rrnL and rrnS). The molecular evidence presented here supports the hypothesis that T. trichiura de M. sylvanus (TMF31) and T. trichiura de P. papio (TPM1) were similar but genetically different with respect to Trichuris sp. from macaques (TMM5). The phylogenetic study also supported the evolution of the different Trichuris species. In conclusion, we suggest the existence of two cryptic species parasitizing M. sylvanus. Citation: Rivero, J.; Callejón, R.; Cutillas, C. Complete Mitochondrial Keywords: complete mitochondrial genome; Trichuris trichiura; phylogeny; whipworms Genome of Trichuris trichiura from Macaca sylvanus and Papio papio. Life 2021, 11, 126. https://doi.org/ 10.3390/life11020126 1. Introduction Academic Editor: Angela Soil-transmitted helminths (STHs) (Ascaris lumbricoides (Ascariasis), Trichuris trichiura Anna Messina (Trichuriasis)) and hookworms (Ancylostomiasis/Necatoriasis) are widely distributed in Received: 9 January 2021 tropical and subtropical areas, with the greatest numbers occurring in sub-Saharan Africa, Accepted: 3 February 2021 the Americas, China, and East Asia [1]. In areas with favorable climatic and environmental Published: 6 February 2021 conditions, poor access to potable water supply, sanitation, and hygiene resources, the transmission of these parasites is higher [2]. Recent data show that about 1.5 billion people Publisher’s Note: MDPI stays neutral are estimated to be affected by STHs worldwide [1]. with regard to jurisdictional claims in Although Trichuriasis is a seriously neglected disease, fortunately, in recent years published maps and institutional affil- hereditary studies are increasing as well as related methodologies that provide new op- iations. portunities for the discovery of novel intervention strategies, with major implications for improving animal and human health and welfare globally [3]. In addition, the implications of genomic studies could also be very relevant in relation to the search for new treatments for immunopathological diseases in humans [4–9]. For example, it has been reported that Copyright: © 2021 by the authors. infections of human patients suffering from immunological disorders (such as Crohn’s Licensee MDPI, Basel, Switzerland. disease) suppress clinical symptoms significantly with pig-Trichuris eggs [6,10–12]. This article is an open access article The nematode mitogenome (complete mitochondrial genome) has several practical distributed under the terms and strengths as a phylogenetic marker, and has yielded well-supported results for clades, conditions of the Creative Commons which were not well resolved using other approaches [13]. The whole genome and tran- Attribution (CC BY) license (https:// scriptome studies have contributed to our understanding of deep node nematode phy- creativecommons.org/licenses/by/ logeny but have been limited by low taxon sampling (which is also biased toward some 4.0/). Life 2021, 11, 126. https://doi.org/10.3390/life11020126 https://www.mdpi.com/journal/life Life 2021, 11, 126 2 of 15 parasitic groups) and by the technical challenges inherent in obtaining a large quantity of genetic information from a single nematode with a small body size. In such cases, the use of mitogenome sequences is one alternative that has been widely applied to many nematode branches where relationships were unclear [13]. Nematode mitogenomes are like those of other animals in many respects, but have a few unusual features including high variation in conservation of gene order across ma- jor branches and the occasional presence of multiple chromosomes [13]. The nematode mitogenome is usually a single, circular molecule ranging in size from 12 to 22 kb and con- taining 36 (sometimes 37) genes—12 (or 13) protein coding genes (PCGs) (cox1–cox3, cytb, nad1-nad6, nad4L, atp6, and rarely atp8), two ribosomal RNA (rRNA) (rrnL and rrnS), and 22 transfer RNA genes (tRNAs). The atp8 gene, which is found in most other metazoan mi- togenomes (except the parasitic Platyhelminthes clade Neodermata) [14], is usually absent in nematode mitogenomes, although it does appear in the order Trichinellida (Trichinella spp. and Trichuris spp.) [13,15–20]. Furthermore, within class Enoplea, members of order Trichinellida (Trichinella spp., Trichuris spp.) show a substantial gene rearrangement even among closely related species, while members of Chromadorea show far less rearrangement in theirs mitogenomes [21]. Kern et al. [13] concluded that the mitochondrial genome is a useful tool for nematode phylogenetic because the diversity within nematode mitogenome architecture, its variable rate of gene rearrangement, and the representation of nearly every kind of lifestyle and habitat ecology within nematodes make this phylum an exciting area for addressing questions about mitogenome evolution. Currently, the complete mt genome of several species of whipworms has been sequenced— Trichuris trichiura from humans and baboons [3,19]; Trichuris sp. from the Endangered François’ Leaf-Monkey [17]; Trichuris rhinopiptheroxella from the endangered golden snub-nosed monkey [20]; Trichuris suis from pig [3,19]; Trichuris ovis from ante- lope [16]; Trichuris discolor from wild yak [16]; Trichuris skrjabini from sheep [22]; and Trichuris muris (LC050561, unpublished). This fact, and considering the hypothesis that Trichuris infecting primates represent a complex of cryptic species with some species capa- ble of infecting both humans and non-human primates (NHPs), reflects the need to delve into the mt genomes of Trichuris spp. of NHPs compared to T. trichiura from humans. Thus, the aim of the present study was to sequence the complete mt genome of Trichuris trichiura isolated from different NHPs. In addition, we carry out structural and phylogenetic comparative analyses with genomes of species of genus Trichuris available in public datasets. 2. Materials and Methods 2.1. Ethics Statement This study does not require approval by an ethics committee. Macaca sylvanus, from which Trichuris specimens were collected from their caeca post-mortem. Trichuris specimens, recovered from the feces of one Papio papio after anthelmintic treatment, were handled and housed in a zoo in strict accordance with good animal practices. 2.2. Parasites, DNA Extraction and Genotyping of Worms Specimen Trichuris worms were recollected from a Guinea baboon (P. papio) at Parque de la Naturaleza de Cabárceno (Cantabria, Spain), from a stool sample after anthelmintic treatment, and a Barbary macaque (M. sylvanus) in Castellar Zoo (Cádiz, Spain), from its caeca post-mortem. Adult Trichuris were recovered and washed in physiological saline, identified morphologically, and then, stored at −20 ◦C until use. Total genomic DNA was isolated from three individual worms according to the manufacturer’s protocol the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Düsseldorf, Germany),used to extract the genomic DNA. Quality of extractions was assessed using 0.8% agarose gel electrophoresis infused with SYBR® Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA) Life 2021, 11, 126 3 of 15 2.3. Mitochondrial Genome Amplification and Sequencing For long-range PCR amplification and next generation sequencing (NGS), different primate derived Trichuris specimens were chosen. One Guinea baboon worm (TPM1) and two Barbary macaque worms (TMF31 and TMM5) were chosen based on their distinct haplotypes identified in previous studies by sequencing the partial gene of mtDNA (cox1, cob and rrnL) and rDNA (ITS1 and ITS2) [23]. To obtain the complete mt genome, firstly, we used the primers designed by Hawash et al. [19] to amplify the Trichuris sp. from baboon (TTB1) and T. trichiura from humans from Uganda (TTHUG) genomes in two overlapping fragments (~8 and ~6 kbp), Nevertheless, we only could amplify the second fragment (~6 kbp), from rrnL to nad1. Then, new primers (MS1F and MS1R) were designed to amplify the other fragment using Primer3 from nad1 to rrnL (http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 1 February 2021)). PCR mix, PCR conditions and PCR primers are summarized in the Supplementary materials (Table S1). The PCR products were checked on SYBR® Safe stained 0.8% Tris-Borate-EDTA (TBE) agarose gels and bands were eluted